miR-142通过上调AKT2的表达促进角膜新生血管形成  

miR-142 promotes corneal neovascularization formation via up-regulating AKT2

作　　者：沈厉 周善璧 毛旖旎 林天兰 岑超 胡雁 侯胜平[3] Shen Li;Zhou Shanbi;Mao Yini;Lin Tianlan;Cen Chao;Hu Yan;Hou Shengping(Department of Ophthalmology, University-town Hospital of Chongqing Medical University;Department of.Ophthalmology,The Sixth People＇s Hospital of Chengdu;Department of Ophthalmology, The First Affiliated Hospital of Chongqing Medical University;Department of Ophthalmology, Chongqing Health Center For Women and Children)

机构地区：[1]重庆医科大学附属大学城医院眼科,重庆401331 [2]成都市第六人民医院眼科,成都610051 [3]重庆医科大学附属第一医院眼科,重庆400016 [4]重庆市妇幼保健医院眼科,重庆400013

出　　处：《重庆医科大学学报》2018年第7期988-993,共6页Journal of Chongqing Medical University

基　　金：重庆市医学科研计划资助项目(编号:2013-1-033)

摘　　要：目的:研究micro RNA142(miR-142)对角膜新生血管形成的影响及其相关机制。方法:实验设置空白对照组、阴性对照组、过表达miR-142组以及干扰miR-142组,分别利用miR-142模拟物(mimic)和抑制物(inhibitor)过表达和干扰miR-142;CCK-8增殖实验检测miR-142对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)增殖能力的影响;双荧光素酶报告基因验证AKT2是否为miR-142的直接靶基因;RT-PCR和Western blot实验检测miR-142对AKT2表达水平的影响。结果:CCK-8增殖实验结果显示空白对照组、阴性对照组和过表达miR-142组96 h的吸光度(absorbance,A)均值分别为872.21±44.20、842.16±49.54和1 407.91±16.58,差异具有统计学意义(F=102.8,P=0.000)。干扰miR-142后HUVECs增殖能力明显减弱(空白对照组=919.69±25.46,阴性对照组=856.76±36.22,干扰miR-142组=602.77±25.62;F=72.8,P=0.000)。双荧光素酶报告基因结果发现过表达miR-142后AKT2表达水平没有明显变化(对照组=0.36,过表达miR-142组=0.46,P=0.147)。RT-PCR结果显示过表达miR-142(0.408±0.046)后AKT2表达水平较阴性对照组(0.173±0.022)升高(P=0.018),干扰miR-142组(0.16±0.02)较阴性对照组(0.362±0.068)明显降低(P=0.037),差异均具有统计学意义。Western blot实验进一步验证了miR-142对AKT2基因的调控作用。进一步研究发现,sh RNA干扰AKT2后miR-142促进HUVECs增殖的作用消失,对照组、过表达miR-142组、过表达miR-142+sh RNA-AKT2组和sh RNA-AKT2组的96 h吸光度值分别为981.80±75.40、1 357.81±47.56、806.39±49.70和921.59±40.38,差异具有统计学意义(F=40.97,P=0.000)。结论:miR-142通过上调AKT2的表达促进HUVECs增殖,从而促进角膜新生血管形成。Objective：To study the impact of miR-142 on corneal neovascularization formation and related mechanism. Methods：Control group,negative group, overexpression miR-142 group and knockdown miR-ld2 group were arranged. Mimic and inhibitor of miR- 142 were used to overexpress or knock down miR-142,respectively. CCK-8 proliferation assay was performed to evaluate the impaction of miR-142 on proliferative ability of human umbilical vein endothelial cells（HUVECs）. Dual-luciferase reporter gene system was used to verify whether AKT2 is the target of miR-142. RT-PCR and Western blot were used to detect effect of miR-142 on AKT2. Results：Results of CCK8 assay showed that absorbance （A） of control group, negative group and overexpression miR-142 group was 872.21 ± 44.20,842.16 ±49.54,1 407.91 ± 16.58, and the results had statistical significances（F=102.8,P=0.000）. Knockdown miR-142 significantly weakened the proliferation rate of HUVECs（eontrol group=919.69 ± 25.46,negative group= 943.43 ± 40.53, knockdown miR-142 group=602.77 ± 25.62, F= 72.8, P=0.000）. Consequence of dual-luciferase report gene system indicated that AKT2 wasn＇ t the target of miR- 142 （control group=0.36, overexpressed miR-142 group=0.46,P=0.147）. RT- PCR assay revealed that expression level of AKT2 was upregulated by overexpressing miR-142（0.408 ± 0.046） than negative group （0.173 ± 0.022） （P=0.018） ; inhibition of miR- 142 blocked the expression of AKT2 （negative group=0.362 ± 0.068, down-regulated miR-142 group=0.160 ± 0.019,P=0.037）. Western blot assay verified the regulation of miR-142 on AKT2. Further study showed that the facilitation of miR-142 on proliferation of HUVECs was thoroughly blocked by shRNA-AKT2,results of control,overexpressed miR-142,overexpressed miR-142＋shRNA-AKT2 and shRNA-142 were 981.80 ± 75.40,1 357.81. ±47.56,806.39 ± 49.70, 921.59 ±40.38,respectively,and the results had statistical significances （F=40.97,P=0.000）. Conclusion ：miR-142 facilitates the proliferation

关 键 词：角膜新生血管 人脐静脉内皮细胞 miR-142 AKT2 

分 类 号：R772.2[医药卫生—眼科]